Method of reducing serum cholesterol level with extract of konjac mannan

ABSTRACT

Water-soluble konjac mannan capable of undergoing gelation when heated in an aqueous alkaline solution is obtained by extracting the ground tuber of the konjac plant with water, separating insoluble matter, dialyzing the solids-free liquid against water and then lyophilizing the dialyzed liquid to remove water. This konjac mannan has utility as a food and exhibits a depressant effect on the serum cholesterol value in warm-blooded animals.

Tlnited States Patent [191 @Egiyama et a1.

[ Dec. 24, 1974 METHOD OF REDUCING SERUM CHOLESTEROL LEVEL WITH EXTRACTOE lKUNJAC MANNAN [75] Inventors: Noboru Sugiyama, Tokyo; IllideoShirnahara, Mihara, both of Japan [73] Assignee: Kabushiki KaishaShimizu Manzo Shoten, Hiroshima-ken, Japan [22] Filed: Sept. 9, 1971[21] App]. No.: 179,201

[30] Foreign Application Priority Data OTHER PUBLICATIONS ChemicalAbstracts: Vol. 60: 4378g (1969); Vol. 57:

l6927h (1962); Vol. 46: 3009i (1952); Vol 36: 65602 (1942); Vol, 32:19502 (1938),

Chemical Abstracts: Vol. 61: 8788g; Vol. 47: 4495c; Vol. 47: 5468h.

Chemical Abstracts: Vol, 36: 70065.

Primary Examiner-Albert T. Meyers Assistant Examiner-Donald B. MoyerAttorney, Agent, or Firm-William J. Daniel [57] ABSTRACT Water-solublekonjac mannan capable of undergoing gelation when heated in an aqueousalkaline solution is obtained by extracting the ground tuber of thekonjac plant with water, separating insoluble matter, dia- Iyzing thesolids-free liquid against water and then lyophilizing the dialyzedliquid to remove water. This konjac mannan has utility as a food andexhibits a depressant effect on the serum cholesterol value inwarm-blooded animals.

1 Claim, N0 Drawings METHOD OF REDUCING SERUM CHOLESTEROL LEVELWll'llI-ll EXTRACT OF KONJAC MANNAN This invention relates to asubstantially pure, watersoluble konjac mannan and to a process forpreparing same. This invention is also directed to a pharmaceuticalcomposition containing this konjac mannan as an active ingredient aswell as to a method of treating warm-blooded animals including humanswith the water-soluble konjac mannan to depress the serum cholesterollevel.

Konjac (Amorphophalus Konjac C. Koch) is a perennial plant belonging tothe family Araceae. Konnyaku, which is made from the tuber of thisplant, has been used traditionally for food in Japan for several hundredyears. The predominant component of edible konnyaku is a glucomannancalled konjac mannan. Edible konnyaku is made from the konjac flour,which is obtained from the dried tuber of this plant.

Several techniques are known in the art for separating konjac mannanfrom konjac flour. In one, konjac flour is boiled in water, treated withFehlings solution to convert the mannan to its copper complex, and thelatter is decomposed again into the mannan after purification, asdisclosed in J. Agr. Chem. Soc. Japan, 6, 991-995 (I930). In another,konjac flour is extracted with water, impurities are removed byprecipitating with ethanol and redissolving the precipitate in waterseveral times, and drying the precipitate finally obtained to obtainpure konjac mannan, as disclosed in Bull. Chem. Soc. Japan, 49, 298-322(1927).

However, konjac mannan obtained by these methods is no longer soluble inwater and is not able to form a konjac gel. This is probably due to akind of denaturation during the processing as has been observed foramylose in alkaline solution, in J. Amer. Chem. Soc., 76, 4595 (I954).

As the result of research on a method of separating konjac mannan insubstantially pure form without serious loss of its valuable naturalproperties, it has been found that a substantially pure, water-solublekonjac mannan can be obtained by extracting the power of the dried tuberof konjac (Amorphophalus Konjac) with water, removing insolublematerials from the extract, dialyzing it against water and thensubjecting the liquid material remaining after such dialysis tolyophilization or freeze-drying.

The water-soluble konjac mannan thus obtained is useful medicinallysince it has been found to have depressing activity on the serumcholesterol level. Since water-insoluble konjac mannan prepared byconventional methods has no such pharmacological activity, it is quitesurprising that the water-soluble konjac mannan prepared by the processdescribed above has a lowering activity on the serum cholesterol level.

Thus, one object of this invention is to provide a substantially pure,water-soluble konjac mannan as a new product.

Another object of this invention is a pharmaceutical compositioncontaining this substantially pure, watersoluble konjac mannan as anactive ingredient.

Still another object of this invention is a method of medically treatingwarm-blooded animals having an elevated serum cholesterol level due, forinstance, to hypertensive symptoms, wherein an effective dose of thesubstantially pure, water-soluble konjac mannan is administered to theanimals.

These and other objects of this invention will become apparent as thedescription proceeds.

According to the process of this invention, konjac flour is mixed withwater under agitation to make a colloidal solution and the resultingsolution is separated from insoluble materials by filtration,centrifugal separation or other suitable procedures, then subjected todialysis treatment and finally to lyophilization to yield substantiallypure konjac mannan.

The konjac flour used as the starting material in this invention isgenerally prepared either by crushing into fine particles the driedtubers of a plant which belongs to the genus Colacacia, preferableAmorphophallus Konjac C. Koch, or by removing starch, fiber, protein,etc., and ethanol-soluble materials from a slurry of ground tubers ofthe stated type. A commercially available konjac flour sold for theproduction of edible konnyaku is also useful as a starting materialwithout further preparatory treatment.

A preferred embodiment of the process of this invention involvesstirring one volume of the konjac flour with 10-200 volumes of water orgrinding one part by weight of raw tuber with 10-100 parts by weight ofwater, whereby the water-soluble components of the kon jac flour, suchas konjac mannan, lower molecular weight impurities and inorganic salts,are dissolved in the water while the water-insoluble components, such asstarch particles, epidermis of tuber and fibrous materials, areprecipitated. The insoluble components are readily removed byfiltration, centrifugal separation or any of the other knownconventional methods. The resulting aqueous colloidal solution is thendialyzed by the usualy methods, for example, through a semipermeablemembrane against tap water for 24-48 hours. By this treatment, thewater-soluble impurities and inorganic materials contaminating thekonjac mannan are removed almost completely.

Finally, removal of water from the resulting solution by lyophilizationor freeze-drying under ordinary conditions leaves a substantially pure,water-soluble konjac mannan as a purely white, cotton-like material,which exhibits the natural properties of konjac mannan, i.e. readysolubility in water and capability of forming konnyaku when heated inthe form of an aqueous colloidal solution with alkali. In contrast, thekonjac mannans prepared by known conventional methods substantially losethese natural properties. Thus, the material of the invention is usefulas a food, i.e. in the preparation of konnyaku.

The conditions used for lyophilization are not critical and can varyaccording to the accepted practice in that art. Temperature in the rangeof --0C and pressures in the range of 0.00l0.l mmHg are acceptible. Thelyophilization need not be carried to substantial dryness if thematerial can by more readily mixed and- /or used in the form of aconcentrated solution.

The substantially pure, water-soluble konjac mannan prepared by thepresent process is a new product not heretofore identified in theliterature and has been found to exhibit a strong serum cholesterollevel de pressing activity, when administered to warm-blooded animals,such as humans, cattle and domestic fowl and the like. Accordingly, thissubstance can be used effec tively to reduce the serum cholesterol valuein the medical treatment of various diseases, such as hypertension inhumans and arterioscolerosis of poultry, in which a high serumcholesterol level is implicated as a causative or aggravating factor.

The substantially pure, water-soluble konjac mannan may be administeredas such, or, if desirable, in the form of a mixture with any carrierconventially used for internal medicaments. When administered to cattleor domestic fowl, the konjac mannan is advantageously incorporated intotheir feed. The dose varies according to the sort of animals but isgenerally administered every day within the range from 0.l g. to g. inseveral portions per kilogram of body weight.

Useful as pharmaceutically acceptable carriers are glucose, lactose, gumarabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc,cone starch, potato starch, keratin, colloidal silica, etc.

The pharmaceutical composition of this invention may be in any desiredform, e.g. powder, tablet, capsule, solution, etc.

This invention will be understood more in detail by reference to thefollowing examples.

EXAMPLE 1 A colloidal solution was prepared by dissolving 0.5 g. of acommercially available konjac flour in 100 ml. of water. After standingfor 2 hours, the solution was filtered through a ISO-mesh nylon filtercloth and then through a glass-filter (304) with suction to remove theinsoluble materials completely. 50 milliliters of the clear filtrate waspacked in a cellophane tube and dialyzed against distilled water for 48hours. The solution remaining after this treatment was transferred to awellcleaned Schale and lyophilized at C. and 0.0lmm pressure for abouthours in a usual manner, Purely white, cotton-like konjac mannan (0.4grams) was thus prepared. Anal. Calcd. for (C H ,O -,'l/8H 0)n; C,43,8371; H, 6.28%; Found: D, 43,8771; H, 6.22%, N,

trace.

The properties of the konjac mannan thus obtained are shown in thefollowing table where those of the starting flour and ofa purifiedkonjac mannan prepared by a known method are also shown for comparison.

Fifteen male rats, each weighing about 100 g., of Wister strain weredivided into three groups and in each group, one rat was fed a basal(normal) diet, another a hypercholesterolemic diet, and the remainingthree rats, three modified versions of the same hypercholesterolemicdiet containing commercially available konjac flour, konjac mannanextracted by alcohol from the konjac flour, and konjac mannan of theinvention, respectively, at a concentration level of 5% by weight.

Fresh food appropriate to each such diet was given ad libitum everymorning. At the end of the test period, the animals were anesthetized byintraperitoneal injection of 5% sodium pentobarbital solution. Blood wastaken by heart puncture into a heparinized syringe and centrifuged.Individual plasma cholesterol levels were measured by the method ofPearson et al.

The results are shown in the following table as the average values ofall groups.

TABLE ll Effect on Hyrx cholesterolemic Activity of Konjac Mannan inRats Plasma Type of Diet Food intake Bvt. gain cholesterol g/8 days 8days in Y ml Normal 134 t 5.9 56% i 2.7 l if) 4.9 Control (hypercholes-132 i 3.2 58.0 i 2.2 l76 9.7 terolemic) As in control with 5% startingkonjac flour i 3.0 45.7 i 2.2 162 t l4.6 (commercially available) As incontrol with 5% alcohol extract from 1 6 i 2.0 56.3 t 2.1 142 I 10.] thekonjac flour As in control with 5% konjac mannan prepared 128 i 1.9 55.]t 3.1 [33 t 5.5

in Ex. I

As is evident from the above table, the konjac mannan prepared accordingto this invention has an excellent lowering activity on the serumcholesterol level in serum.

According to conventional dialysis procedure, such as is employed in thepractice of the invention, the concentration of the solution is usuallywithin the range of 0.l-1.0% by weight, preferably 0.34.071 by weight.The dialysis temperature is generally within the range of O-30C,preferably 3l()C. Above about 30C, the

solution tends to undergo decay where the circumstances are suitable forgrowth of microorganisms. Utilizable as semi-permeable membranes arepreferably cellulosic materials, especially cellophane marketed byVisking. Membranes of animal origin, for example, urinary bladdermembranes, are generally inappropriate for the purpose of this inventionfor the reason that their pore size is too large. By employing membranesof vegetable origin such as cellophane, particulate impurities areremoved to yield a product having a definite size of molecule. Theconditions of the dialysis are not especially critical in practice ofthis invention, however, and any ranges would be suitable here.

As mentioned above, the product of this invention can also be utilizedas a food or food additive. Ordinary edible konjac is colored slightlygray brown to light brown and contains insoluble black residual matter.In contrast, the product of this invention is highly purified konjacmannan which is colorless to white and entirely soluble in water. Thus,the product of this invention is useful as purified edible konjac or afood additive, for example, a thickening agent for jelly. As ordinaryedible konjac is colored more or less, it has little or no LII UNITEDSTATES PATENT OFFICE CERTIFICATE OF CORRETION Patent No. 3,856,945 d 1December 24, 1974 Inventor(s) I 'Noboru SUGIYAMA et al It is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

In the Heading of the Patent under Foreign Application Priority Data,correct the application No. from "45-12828" to Signed and sealed this11th day of March 1975.

(S Attest: C. MARSHALL DANN Commissioner of Patents RUTH C. MASON andTrademarks Attesting Officer

1. A METHOD FOR REDUCING THE SERUM CHLOESTEROL LEVELS INHYPERCHOLESTEREMIC WARM-BLOODED ANIMALS WHICH COMPRISES ORALLYADMINISTERING A THERAPEUTICALLY EFFECTIVE DOSE OF SUBSTANTIALLY PURE,WATER-SOLUBLE KANJAC MANNAN IN THE RANGE OF 0.1-10 G. PER KG OF ANIMALBODY WEIGHT PER DAY, SAID KONJAC MANNAN BEING OBTAINED BY EXTRACTINGWITH WATER THE GROUND TUBER OF THE KONJAC PLANT, DIALYZING SUCH EXTRACTAGAINST WATER AND LYOPHILIZING THE THUS DIALYZED EXTRACT TO REMOVEWATER.